Emergence of population heterogeneity in Klebsiella pneumoniae with a blaOXA-232-harboring plasmid: carbapenem resistance, virulence, and fitness

Abstract

Background: This study aimed to investigate the population heterogeneity on carbapenem susceptibility in Klebsiella pneumoniae strains that acquired a blaOXA-232-bearing ColE-type plasmid. Methods: A blaOXA-232-bearing plasmid was electroporated into two carbapenem-susceptible K. pneumoniae strains. High- and low-carbapenem-resistant subpopulations were identified and isolated using patch plating. The strains were subsequently subcultured in antibiotic-free media, yielding two distinct populations: a stable, high-level carbapenem-resistant strains and a heterogeneous strains. Antibiotic susceptibility tests, time-killing assays, and population profiles were conducted, along with a competition assay was performed and the growth curve analysis. To assess virulence, we performed human serum resistance and Galleria mellonella infection assays, and measured the expression of virulence genes using qRT-PCR. Additionally, whole genome sequencing was carried out for further anaysis. Results: Introduction of pOXA-232 into carbapenem-susceptible K. pneumoniae strains resulted in two isogenic transformants with distinct resistance profiles: an unstable, high-level carbapenem-resistant (HCR), and highly virulent subpopulation; and a stable, low-level carbapenem-resistant (LCR), and low-virulence subpopulation. Whole genome and expression analyses revealed dysfunctionality of ompK36 in HCR subpopulations. Subculturing of HCR led to the re-emergence of heterogeneous populations with variations in carbapenem resistance and an additional compensatory mutation of 9,000 bp deletion in the genome. Thus, stable HCR strains featuring both mutations in ompK36 and compensatory mutations developed. Conclusion: This study demonstrated that underlying heterogeneity can promote the emergence of stable, high-level antibiotic resistance, even with the introduction of a plasmid carrying a low-level antibiotic resistance gene, such as blaOXA-232. This highlights the critical need to closely monitor bacterial population dynamics.